Recently, it has been found that purified cell walls separated from microorganisms such a BCG (Bacillus Calmette-Guerin) or Nocardia, etc. have a host-mediated antitumor effect [Journal of National Cancer Institute, Volume 52, page 1571 (1974); Gann, Volume 69, page 19 (1978); Gann, Volume 67, page 669 (1976); Cancer Immunology and Immunotherapy, Volume 4, page 95 (1978)], and studies on manufacture of an antitumor agent from cell walls of other species have been made to separate purified cell walls of microorganisms having a higher antitumor activity and a clinical applicability. Particularly, gram-positive bacteria widely distributed in the natural world with no fear of existence of endotoxins have been regarded as very important, and purified cell walls having an antitumor effect have been obtained from some of gram-positive bacteria [Proceedings of the Japanese Cancer Association, the 40th Annual Meeting, page 107 (1981)].
Cell walls of gram-positive bacteria have an envelope-like structure based on a covalent bond of peptidoglycan with the so-called special structural substance which is specific to the species of bacteria in question and contains polysaccharides or teichoic acids as the main ingredient. The cell walls locate in contact with the outside of cell membrane to keep the shape of cells and act to protect the cytoplasm and its function from various external stimuli. Particularly, the peptidoglycan moiety is not only important as an active center of antitumor effect, but plays an important role also to keep the entire shapes of cell walls. That is, peptidoglycan is composed of reticular binding of glycan chains consisting of N-acetylglucoasamine and N-acetylmuramic acid with peptide chains in a three-dimensional structure and form a water-insoluble, physically very rigid macropolymers.
According to the conventional process for separating and extracting purified cell walls from bacteria, intracellular cytoplasmic ingredients were liberated and removed under specific conditions at first, and then crude cell walls remaining as water-insoluble residues were separated, and subjected to repeated purification treatment with enzymes such as protease and nuclease. Among these series of operations, particularly liberation and removal of intracellular cytoplasm ingredients were very important for increasing chemical purity of purified cell walls, and its specific procedures based on the species of bacteria have been widely studied.
That is, the studies are varied with the species of bacteria, including, for example, relatively easy occurrence of liberation of cytoplasmic ingredients only by changes in osmotic pressure as in vibrio costicolus as a gram-positive bacteria ( Journal of General Microbiology, Volume 20, page 32 (1959)], or by treatment with a surfactant as in Escherichia coli [Journal of Bacteriology, Volume 136, page 723 (1978)], and only possible removal of cytoplasmic ingredients by disruption of cell walls by very vigorous physical treatment as in gram-positive coccus [Biken's Journal, Volume 2, page 143 (1959)], and such variety depends on physical and chemical properties of cell walls in the bacteria in question.
Generally, in the case of gram-positive bacteria, it is indispensable to disrupt cell walls by a physical means such as ultrasonic oscillation etc. or by a chemical means such as acids or cell wall-lytic enzymes, etc. owing to the said complicated ingredients and physically rigid cell walls, and such destructive treatment has been so far applied to many gram-positive bacteria as a common means for isolating purified cell walls of gram-positive bacteria. Thus, there has been no technical concept of extracting only purified cell walls from gram-positive bacteria without subjecting the cell walls to a physical and/or chemical destructive treatment.
However, according to the conventional process using the destructive treatment, not only cell walls are cut into finer fragments, resulting in considerable reduction in yield, but also non-specific breaking of the covalent bonds takes place on the peptidoglycan moiety and this leads to a very great disadvantage of lowering the antitumor effect from the bifidobacteria to improve the said conventional process, and have established the present invention.
The present invention provides an antitumor agent characterized by cell walls of intact cells of bifidobacteria, intracellular ingredients being removed from the cells without subjecting the cell walls to physical and chemical disruption, thereby leaving the cell walls, and also provides a process for preparing an antitumor agent, characterized by treating intact cells of bifidobacteria with a surfactant, and then treating the cells with proteases, nucleases, an organic solvents and a dilute acid, thereby removing intracellular ingredients from the cells and extracting the cell walls without disrupting the cell walls of the cells.
An object of the present invention is to provide a substance having antitumor activity comprising cell walls of cells of bifidobacteria and having a high activity.
Another object of the present invention is to provide a process for manufacturing a substance having antitumor activity having a high activity from bifidobacteria in high yield.